Project Abstract The protozoan parasite Cryptosporidium is a leading cause of diarrheal disease (cryptosporidiosis) and death in young children and immunocompromised individuals. Currently, there are no vaccines or effective drugs to prevent or treat cryptosporidiosis. Our understanding of the interactions of Cryptosporidium with its host cell is very limited. Cryptosporidium has a unique mode of invasion that differs from other related apicomplexan parasites. The parasite creates an ‘intracellular but extracytoplasmic’ niche where it sits on top of the intestinal epithelial cell, engulfed by the host cell membrane, and is separated from the host cell cytoplasm. A key feature of Cryptosporidium invasion is the rapid polymerization of host actin and cell remodeling, which leads to the formation of the trophozoite with an ‘actin pedestal’ underneath at the host-parasite interface. Although Cryptosporidium releases hundreds of secretory effector proteins from four organelles—rhoptries, micronemes, dense granules, and the newly discovered ‘small granules’—the role of these proteins in host-parasite interactions remains largely unknown. Notably, the parasite effectors that manipulate the host machinery to promote actin polymerization during invasion are not known. To address this knowledge gap, we have identified Cryptosporidium secretory proteins that are potentially involved in manipulating host actin polymerization machinery during invasion for the parasite to establish its unique niche. The goal of this exploratory proposal is to utilize a combination of molecular genetics and cell biology approaches, and animal infection experiments to investigate the role of the top identified candidates and their interacting host proteins in promoting actin polymerization. The results of this study will improve our understanding of Cryptosporidium biology and host- parasite interactions. The new knowledge gained from this work will support future development of vaccines and effect